The complex diterpene pseudoalkaloid paclitaxel is the most important antineoplastic agent discovered in the last 20 years. It is present in the plant tissue of the family Taxaceae, especially in several species of Yew trees and shrubs that comprise the genus Taxus. While many of the taxanes possess cytotoxic properties making them useful for therapeutic treatment of cancers, paclitaxel has been the subject of the greatest study and was the first to obtain regulatory approval for clinical use.
Regulatory standards for pharmaceutical formulations of natural products isolated from plant sources are highly demanding in terms of reliability of production methods and purity of the sample obtained, especially where (as with paclitaxel) the source material contains other cytotoxic compounds of similar structure and biological activity. Extracts of Yew plants typically contain several taxanes including, for example, baccatin III, cephalomannine, 10-deacetyl taxol ("10-DAT"), 10-deacetyl baccatin III, 7-epi-10-deacetyl taxol ("7-epi-10-DAT"), 7-epi-paclitaxel and taxol-C. Such compounds are difficult to separate from paclitaxel on an economical scale. The desired paclitaxel composition for human use requires isolation methodologies that reproducibly yield paclitaxel preparations containing at least 98.5% with low levels of taxane impurities. Furthermore, regulatory standards demand very low levels of residual solvents, such as methanol, hexane and acetone, which are typically used in the extraction of paclitaxel.
In addition to meeting regulatory requirements for reproducibility and purity, economic considerations demand that paclitaxel isolation methodologies be as simple and inexpensive as possible. Taxol is present in very low amounts in Yew plants. Thus, even when using tissue having a relatively high paclitaxel content, such as the bark of T brevifolia, and T. yunnanensis trees (about 0.02%) or the needles and roots of T. x media Hicksii shrubs (0.005-0.15%), very large amounts of paclitaxel-containing material must be harvested to obtain relatively small amounts of paclitaxel. This economic cost is exacerbated by the fact that conventional protocols for extraction and purification of paclitaxel are inefficient in terms of yield and/or require expensive materials and equipment, making industrial scale preparation of pharmaceutical grade paclitaxel extremely expensive. Procedures that rely on less inexpensive materials, such as solvent extractions followed by normal phase silica columns, typically result in paclitaxel preparations of low purity, low yield or both. Alternative procedures for improving yield and purity often rely on methods that are impractical and/or expensive on an industrial scale.
A large scale operation requires a robust procedure that is simple, inexpensive, reproducible, and provides a high yield and high level of purity. High yield and high purity are often at odds. High yield in a multistep procedure ideally requires high recovery for each intermediate step. Unfortunately, high recovery obtained by solvent extractions and partitions of paclitaxel often yields bulk fractions containing large amounts of impurities, especially those with related structures. To obtain high purity from these bulk fractions requires using chromatographic procedures that can resolve closely related components. However, chromatography media such as silica, which is relatively inexpensive and can be disposed of after use (thus avoiding regeneration), does not have effective resolving power if the feed materials are not previously enriched for paclitaxel. This often requires use of intervening steps that improves purity but sacrifices yield.
Large scale isolation of paclitaxel from more than 8000 lbs of bark by use of solvent extractions, differential crystallization and normal phase silica chromatography was described by Huang et al., (Journal of Natural Products 49(4): 665-669, 1986). This method demonstrated the general solubility and chromatographic properties of paclitaxel, and illustrated the difficulty encountered in obtaining a good yield of highly purified material using conventional techniques. Most subsequent techniques are essentially modifications of the Huang et al. method that have shown increased yields and/or purity. However, these methods still fall short of providing a satisfactory process for large scale isolation. Furthermore, most of the improved methods are based on laboratory scale experimentation that poorly translates into an efficient large scale production.
For example, the laboratory scale procedures described in U.S. Pat. Nos. 5,279,949 and 5,478,736 to Nair (also PCT Publication No. WO 97/09443) use activated charcoal for decolorizing an initial 70% ethanol/water extract and early filtration through Celite. The decolorized extract is subsequently extracted with ethyl acetate, and evaporated to precipitate taxanes. The taxanes are redissolved in ethyl acetate and loaded onto a first silica column that is eluted with a hexane/ethyl acetate gradient, and further purified by tandem silica columns or, alternatively, by reverse phase chromatography. Unfortunately, the procedure of Nair is not amenable to industrial scale isolations for several reasons. First, the amount of charcoal required (up to about 5%-15% w/v extract) creates processing, cleanup and flow problems when used on an industrial scale. In addition, neither the charcoal nor subsequent steps aid in removing key impurities that are difficult to separate and remain as persistent impurities even after purification over tandem silica columns. As a result, the final product obtained using three normal phase silica chromatography steps is not sufficiently pure for pharmaceutical preparations. Therefore HPLC and/or reverse phase chromatography is further recommended, which inevitably results in a lower yield of material and significantly increases the cost. Overall, the laboratory scale process described by Nair is extremely impractical and expensive on large scale.
U.S. Pat. Nos. 5,618,538 and 5,480,639 to ElSohly et al. provide several laboratory-scale examples related to isolating paclitaxel and other taxanes that include evaluation of a variety of plant species, plant parts, solvent systems for initial extraction, mechanical methods for extraction, and solvents for paclitaxel partitioning. The crude solvent extracts obtained are further subjected to Celite absorption followed by three normal phase silica chromatography steps using hexane/acetone, methanol/methylenechloride, and ethylacetate/methylenechloride as carrier solvents. The eluates from column fractions are evaporated before proceeding to the next step, and are not subject to intervening precipitation or crystallization steps to enhance purity. A final yield of 26 mg of paclitaxel described as "pure" by HPLC analysis was obtained from 500 g of T media "dark green spreader" leaves having a starting paclitaxel content of 0.0074%. Thus, this procedure represents a method for recovery of about 70% of the paclitaxel present in the initial plant material. However, although the procedure provides a high yield of paclitaxel, it requires up to four repeated chromatographic steps. In addition, the use of large amounts of celite, silica and solvents relative to other methods is a significant hindrance for implementing this process on large scale.
The procedures described in U.S. Pat. Nos. 5,380,916, 5,475,120, and 5,670,673 to Rao (also PCT Publication No. WO 92/07842) use a series of solvent extractions employing ethanol, chloroform, ligroin, benzene and methanol followed by reverse phase chromatography exemplified using HPLC with an acetonitrile eluent. However, reverse-phase chromatography is not practical for large scale preparation of paclitaxel primarily because plant extracts from Yew contain numerous substances that interfere with column performance and maintenance. Furthermore, reverse-phase chromatography media is expensive and not easily regenerated, thus creating high replacement costs, and large scale production based on an acetonitrile eluent is not practical unless total containment is provided, which is very costly. Rao reports a yield of about 0.05% of the biomass as paclitaxel, but still further refinement is needed to obtain pharmaceutical grade paclitaxel. As a general principle, only those processes shown to produce high purity paclitaxel on a very large scale are suitable candidates for industrial production, because large scale generally has an adverse effect on product quality compared to the same process executed on small scale.
The procedure described in PCT Publication No. WO 96/34973 by hong et al. involves use of a preliminary treatment of a methanol/dichloromethane extracts with a synthetic absorbent, such as activated charcoal or clay, followed by multiple dichloromethane washings and precipitations, first using hexane to produce a 23% paclitaxel precipitate, then using multiple alcohol/water fractional precipitations to produce an 85% extract suitable for final purification of paclitaxel by HPLC. This procedure is also unsuitable for large scale preparation, not only because of the use of activated charcoal having the drawbacks indicated above, but because hexane and alcohol/water precipitation steps have been found to give irreproducible results. In addition, the procedure is inefficient and costly because of the time and materials required for multiple precipitation steps (each requiring several days and large quantities of solvent), and the expense of HPLC techniques applied on an industrial scale.
It should be noted that Hong et al., in the abstract, refer to "a high recovery of over 90%", but a closer examination reveals that this recovery rate is characteristic for only the best step yields and that the overall yield is much lower. Hong et al. report cases of step yields at 100%. Such quantitative yields can sometimes be obtained in the laboratory by the use of relative large quantities of auxiliary materials, the application of very costly and effective techniques (such as HPLC), or the use of time consuming and/or labor intensive methods. All these aspects are apparent in the process described by Hong et al., thus emphasizing that, while the procedure may be suitable for laboratory practice, it is of very limited utility for large scale production.
The procedure described in PCT Publication No. WO 98/07712 by Zamir uses an organic extraction followed by an aqueous wash, charcoal treatment, and precipitation/recrystallization of taxanes in the presence of non-polar solvents such as toluene and ether. Finally, a recrystallized fraction is subjected to either reverse phase chromatography or HPLC (or both) to separate a variety of taxanes including paclitaxel. This procedure has many of the drawbacks mentioned above, including the use of charcoal (which requires an additional cleaning step to remove fine charcoal particles), and the use of expensive reverse phase and HPLC systems to obtain paclitaxel of sufficient purity. In addition, the extensive use of methylene chloride and acetonitrile throughout the process requires expensive containment.
The procedure described in Chinese Application No. 96102442.9 by Liu and Yang relies on the use of porous polymer reverse-phase chromatography early in an extraction process. An alcohol extract is partitioned into dichloromethane, subjected to an aqueous wash, filtered and immediately loaded onto a porous polystyrene reverse-phase column which is eluted with increasing steps of methanol/water. A paclitaxel fraction eluted from the column containing 50-68% paclitaxel contained about 10% cephalomannine, and a fraction containing 60% cephalomannine contained about 10% paclitaxel. Another fraction containing 30% 10-deacetyl baccatin III may be precipitated in acetone and water to obtain 76% 10-deacetyl baccatin-III. Further purification of paclitaxel required an additional reverse phase step to yield a fraction containing about 98% paclitaxel. However, this is not sufficiently pure for pharmaceutical purposes, especially when the remaining 2% impurities likely contain cephalomannine.
The procedures described in U.S. Pat. Nos. 5,393,896 and 5,736,366 to Margraff and U.S. Pat. Nos. 5,453,521 and 5,393,895 to Gaullier et al. are related to the isolation of 10-deacetyl baccatin III which is useful as a precursor for chemical synthesis of paclitaxel. The processes described therein are specifically directed to isolation of the precursor molecule using alcohol extraction of paclitaxel-containing material, an aqueous wash, organic solvent extraction, and selective precipitation. The methods are not instructive for the isolation of paclitaxel, but do illustrate the difficulty encountered in separating taxane compounds obtained from extracts of Yew.
The procedure described by Shibuya in European Pat. Publication No. 700 910-A1 relates to extracting paclitaxel from T. sumatrana, using solvent extraction, liquid-liquid partitioning, silica chromatography, Sephadex chromatography and reverse phase HPLC to obtain a paclitaxel yield of only 0.006%. The low yield again illustrates the difficulty in creating methodologies for the efficient extraction of paclitaxel from plant material using reverse phase and HPLC techniques. Again, this method is very impractical and expensive for large scale production.
The procedure of U.S. Pat. No. 5,744,333 and European Patent No. 553,780 B1 to Cociancich and Pace provides a method of isolating paclitaxel and related compounds from tissues of ornamental Taxus plants, such as T. x media Hicksii or T. cuspidata and cultures prepared therefrom. The steps include making a methanol extract of the plant material, vacuum drying the methanol extract, performing a liquid-liquid extraction with cyclohexane and methylenechloride, followed by silica gel HPLC chromatography to obtain purified paclitaxel, though not of pharmaceutical grade. Similarly Chinese Application No. 94114041.5 by Lu et al. describes a general method for obtaining a 0.007% yield of paclitaxel having greater than 98% purity from T. floridana or T. mairei, using solvent extraction followed by reverse phase silica chromatography or reverse phase HPLC. Again, the drawbacks of HPLC and reverse phase chromatography as employed in these references have been mentioned above.
Accordingly, there is a need in the art for paclitaxel purification methods that can be used on a very large scale, that are rapid, that employ conventional low to medium pressure normal-phase chromatography, that use inexpensive materials, and that provide reproducible isolation of a paclitaxel composition having pharmaceutical grade purity and a high yield. The present invention fulfills these needs and provides further related advantages.